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Acta Crystallographica Section D
Biological
Crystallography
JLigand: a graphical tool for the CCP4
template-restraint library
ISSN 0907-4449
Andrey A. Lebedev,a* Paul
Young,b Michail N. Isupov,c
Olga V. Moroz,d Alexey A.
Vagind and Garib N. Murshudove
a
CCP4, STFC Rutherford Appleton Laboratory,
Harwell Oxford, Didcot OX11 0QX, England,
b
York Digital Library, University of York,
Heslington, York YO10 5DD, England,
c
Henry Wellcome Building for Biocatalysis,
Biosciences, College of Life and Environmental
Sciences, University of Exeter, Stocker Road,
Exeter EX4 4QD, England, dStructural Biology
Laboratory, University of York, Heslington,
York YO10 5DD, England, and eStructural
Studies Division, MRC Laboratory of Molecular
Biology, Hills Road, Cambridge CB2 0QH,
England
Biological macromolecules are polymers and therefore the
restraints for macromolecular refinement can be subdivided
into two sets: restraints that are applied to atoms that all
belong to the same monomer and restraints that are associated
with the covalent bonds between monomers. The CCP4
template-restraint library contains three types of data entries
defining template restraints: descriptions of monomers and
their modifications, both used for intramonomer restraints,
and descriptions of links for intermonomer restraints. The
library provides generic descriptions of modifications and
links for protein, DNA and RNA chains, and for some
post-translational modifications including glycosylation.
Structure-specific template restraints can be defined in a
user’s additional restraint library. Here, JLigand, a new CCP4
graphical interface to LibCheck and REFMAC that has been
developed to manage the user’s library and generate new
monomer entries is described, as well as new entries for links
and associated modifications.
Received 22 November 2011
Accepted 19 January 2012
Correspondence e-mail:
[email protected]
1. CCP4 library of template restraints
1.1. Overview
In the early days of the CCP4 project (Collaborative
Computational Project, Number 4, 1994; Winn et al., 2011), the
least-squares minimization program PROLSQ (Hendrickson
& Konnert, 1980) was adopted as the main macromolecular
refinement tool within the project. PROLSQ required a standalone program PROTIN which analyzed the atomic coordinate data and identified the atoms and ideal geometric values
involved in the individual stereochemical restraints for the
refinement. The ideal geometric values for amino acids and
some ligand compounds were stored in the library file
protin.dic. Every time a user needed to create a description
of a novel ligand he had to manually prepare a list of its ideal
values in PROTIN dictionary format with the help of the
program MAKEDICT (Collaborative Computational Project,
Number 4, 1994) and append it to the dictionary. The
PROTIN dictionary was extensively modified at the beginning
of the 1990s to conform to the Engh and Huber parameters
(Engh & Huber, 1991). Earlier versions of the maximumlikelihood refinement program REFMAC (Murshudov et al.,
1997) also used PROTIN for preparation of stereochemical
restraints. However, the limitations of the PROTIN dictionary
incited the REFMAC developers to give up using PROTIN
and to design a more flexible library of template restraints and
incorporate restraints preparation into REFMAC.
The CCP4 library of template restraints (further referred
to as the restraint library, but also known as the monomer
library; Vagin et al., 2004) originates from the library which
Acta Cryst. (2012). D68, 431–440
doi:10.1107/S090744491200251X
431
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was developed for the Protein Data Bank (PDB; Bernstein
et al., 1977) in order to store descriptions of ligands found in
macromolecular structures. Since then, the restraint library
and its PDB analogue the Chemical Components Dictionary
(Dimitropoulos et al., 2006) have diverged, although both use
the same CIF format and have some common or similar entry
types. The Chemical Components Dictionary is updated
concurrently with the release of each coordinate entry
containing a new ligand, while the CCP4 restraint library is
updated less frequently: only with the main releases of the
CCP4 suite. Additionally, the restraint library contains all
of the information required for macromolecular structure
refinement with REFMAC (Murshudov et al., 2011). Along
with descriptions of atoms and bond orders, it stores information about covalent bond lengths and angles, torsion angles
and lists of chiral centres and planar groups. Another important feature of the restraint library is that it also provides
definitions of restraints for linkages between the monomers in
polymers. While the restraint library was originally developed
for REFMAC, the principles of organization and format of
the library were subsquently adapted for use in PHENIX
(Moriarty et al., 2009) and BUSTER (Bricogne et al., 2011).
The library is also used in Coot (Emsley et al., 2010) for input
of data required for real-space refinement of ligands and in
CCP4mg (Potterton et al., 2004).
The restraint library contains three main types of entries:
monomers, modifications and links. Monomer entries define
the molecular graph, atom names and template restraints for
isolated compounds, including restraints for bonds, angles,
torsion angles, planes and definitions of chiral centres. Modification and link entries are required to generate restraints for
modified monomers and polymers and are discussed in more
detail in the next section. There are also several auxiliary
entries, including one defining atom energy types used by the
program LibCheck (Vagin et al., 1998, 2004) to generate new
descriptions of monomers.
Currently, the program Sketcher, which is part of the CCP4i
interface (Potterton et al., 2003), is used as a graphical interface for LibCheck. While Sketcher is well adapted for
generation of new monomer entries from scratch or from
existing monomer entries, it is not possible to use it for the
creation of link descriptions. Therefore, JLigand, a more
versatile graphical interface for LibCheck, has been developed.
following scheme.
Similarly, the generic peptide modification ‘NH1’ describes the
deletion of an H atom and a proton from a protonated amino
group.
At this point, the generic link ‘TRANS’ can be applied to two
modified peptides to generate template restraints for the
covalent linkage between them.
The mechanism described above may look as if it has
an unnecessary complexity. It can be demonstrated why it
nevertheless makes sense. Let us consider two approaches. In
the first, only one type of data is available: the monomers. Let
us exclude proline, which is a special case, and consider 20
different amino acids with an identical structure of the main
chain (including selenomethionine). To describe all possible
positions of the residue (N-terminal or C-terminal or in the
middle of the chain) and major conformations (cis and trans)
six different versions of each amino acid would be required,
and this would amount to a total of 120 data blocks in the
library. Such an approach does not require any additional type
of data because the restraints associated with the covalent
linkage between two amino acids are added to the description
of one of them.
In the second approach, which is adopted in the restraint
library, each amino acid is defined only once,1
while the covalent linkages between amino acids,
1.2. Covalent modifications and linkages
are defined separately using four library entries: two modifications
Template restraints for covalent linkages between monomers are defined in the restraint library using modification and
link library entries. This mechanism was developed to describe
changes resulting from chemical reactions, including polymerization reactions. In particular, modifications describe
changes in monomers, while links allow joining of the modified
monomers together. For example, a generic modification
‘DEL-OXT’ describes the deletion of carboxyl atom OXT
from an amino acid and updates geometrical restraints around
the deleted atom. This modification can be represented by the
1
In the restraint library, 21 ‘standard’ amino acids are described as being
bound in the middle of a polypeptide chain, without OXT, HN1 and HN2
atoms but with HN atoms. This is reverted to a description of free unbound
amino acids when the library is being read. All the ‘special’ amino acids (see
x2.2) as well as nucleotides and sugars are presented by descriptions of their
free forms. The ‘standard’ amino acids are kept in their polymerized form for
compatibility reasons and can be reverted to their free forms straight away
with no changes in the REFMAC code.
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Acta Cryst. (2012). D68, 431–440
research papers
and two links,2
Such an approach requires two additional types of library
entries. However, it substantially reduces the redundancy of
the data stored in the library and, in our example, requires
only 24 data entries instead of 120 for the description of all
possible polypeptides containing the 20 amino acids.
Both links in the above example are generic from either
side, i.e. neither the first nor the second monomer related by
the link has to be a specific monomer, it only has to belong to a
certain group (the group ‘peptide’ in this example). There are
similar generic links for DNA and RNA polymers and also
generic links for sugars that will be discussed in more detail
later. Along with the generic links, there are also links that are
generic from only one side. These include links associated with
the peptide bonds between proline and other amino acids
(PTRANS, NMTRANS, PCIS, NMCIS) and some of the
terminal modifications of amino acids (e.g. NME_N-C for
amidation of the C-terminus and FOR_C-N and ACE_C-N for
formylation and acetylation of the N-terminus).3 This group
also includes links for members of the group ‘pyranose’ with
specific amino acids: Asn, Ser or Thr (NAG-ASN, NAG-SER
and NAG-THR, respectively). There are also specialized links
which can only be applied to two particular monomers,
including one describing S—S bridges between cysteines.
Overall, the restraint library contains link and modification
data for (i) polypeptide chains, (ii) S—S bridges, (iii) polynucleotide chains and (iv) glycosylated proteins. In addition,
the restraint library contains modifications which are applied
on their own and not in a context of any link for (i) some of the
terminal peptides and nucleotides, (ii) methylated nucleotides
and (iii) deprotonated states of phosphate and carboxyl
groups. More details on the monomer library can be found on
the CCP4 website in the LibCheck documentation.4
DEL-HO2, DEL-HO3, DEL-HO4 or DEL-HO6 (deletion of
an H atom), to make a corresponding link ALPHA1-2,
ALPHA1-3, ALPHA1-4, ALPHA1-6 or BETA1-2, BETA1-3,
BETA1-4, BETA1-6 with the first modified monomer.
Thus, for typical cases of glycosylation all the necessary
modifications and links are present in the restraint library and
by default REFMAC uses these library descriptions with no
need for any additional instructions. An example of such a
case is presented in Fig. 1. This is a crystal structure of mutant
butyrylcholinesterase (Nachon et al., 2011; PDB entry 2xmb)
containing a simple branching polysaccharide with three
residues. At this point it has to be mentioned that the PDB
header LINK record conventions differ between the PDB and
REFMAC. That from the PDB header lacks any reference
to the link library entry. Therefore, if a PDB file has been
downloaded from the PDB with the purpose of validation or
electron-density analysis and the structure is to be re-refined
using REFMAC, it is highly likely that REFMAC would assign
the link type incorrectly, e.g. with incorrect target chiralities of
the involved atoms. The easiest way to avoid this is removal of
glycosylation-related LINK records from the file header (or
removal of the whole header except for the CRYST1 record).
A more common scenario is building a polysaccharide into
experimental electron density. It is always good practice to add
sugar monomers one at a time and make sure that REFMAC
has interpreted the user’s intention correctly. One possible
problem could be the absence of the required link descriptions
in the restraint library when the polysaccharide to be built is
somehow special. In such a case the missing link can be
defined by the user as described in x2. The main checkpoints
are as follows: (i) REFMAC terminates normally, without
error messages; (ii) the header of the output PDB file contains
all necessary link records in REFMAC format; and (iii) the
1.3. Generic links for sugars
The modification and link mechanism is even more important for the description of covalent linkages between sugar
monomers. The number of possible combinations of these
monomers is larger than that for amino acids. Each sugar
monomer has more than two atoms through which links to
other sugar molecules can be made. For example, in v.5.28 of
the library there is a group of compounds called ‘pyranose’
(207 members) and any monomer of the group can undergo
modification DEL-O1 (deletion of the O1 and HO1 atoms).
Another sugar moiety can be modified by one of the following,
2
The modifications DEL-HN1 and DEL-OXT required for TRANS or CIS
links are applied based on the position of the amino acid in the polypeptide
chain, i.e. are applied if the chain contains residues with smaller or larger
sequence number, respectively. Therefore, TRANS, CIS and several other
links do not contain references to these modifications.
3
Some monomer entries represent groups rather than free compounds and do
not require further modification for their incorporation into a polymer chain,
such as, for example, monomer entry FOR representing the formyl group,
which can terminate the polypeptide chain.
4
See also http://www.ysbl.york.ac.uk/~alexei/dictionary.html.
Acta Cryst. (2012). D68, 431–440
Figure 1
Example of glycosylation, which only requires the generic links for sugars
defined in REFMAC’s standard monomer library for refinement. The
figure shows one molecule of -l-fucose (FUL) and two molecules of
N-acetyl-d-glucosamine (NAG) forming a polysaccharide covalently
bound to Asn241 in the crystal structure of the G117H mutant of human
butyrylcholinesterase (Nachon et al., 2011; PDB entry 2xmb). Refinement
of such a structure will implicitly use the links NAG-ASN (1), BETA1-4
(2), ALPHA1-6 (3) and associated modification from the restraint library.
This figure and Fig. 3 were generated using CCP4mg (Potterton et al.,
2004).
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log file contains warnings saying that, for example, link
‘ALPHA1-6’ is found (Fig. 2). Such warnings prompt the user
to check whether the correct types of links have automatically
been assigned. Indeed, if sugar monomer(s) have not been
very accurately positioned in the electron density then it is
quite likely that REFMAC will assign an incorrect type of link
between the new sugar monomer and its neighbour. In this
case REFMAC will terminate normally. It will however write
the incorrect type of link into the output PDB-file header
and therefore the mistake will persist during further model
building and refinement. In such a case the following checkpoints apply. Assignment of a correct link type means that (iv)
the log file contains no warnings about significant deviation
from the target chirality of sugar links and (v) no significant
peaks are observed in the Fo Fc-type difference map near
the linked atoms. If things do go wrong then changing the link
type in the PDB-file header may help (e.g. from ALPHA1-3 to
BETA1-3). Otherwise, more careful fitting of monomers into
the electron density will be required or perhaps the choice of
different sugar monomers.
2. User’s additional library of restraints
The (standard) restraint library is based on the PDB Chemical
Components Dictionary that only contains the ligands found
in deposited macromolecular structures and normally users
do not have permissions to make modifications to it. Any new
chemical features are to be stored in an additional user’s
library. The two libraries are similar in that both may contain
monomer, modification and link entries. They can be used in
refinement simultaneously. In this case, the entries from the
additional library take precedence over entries that have the
same name in the standard library. This behaviour makes it
possible, for example, to use updated versions of ligands.
The objective of JLigand is to generate or update the
additional library.
2.1. Generation of template restraints for isolated ligands
using JLigand
2.1.1. Minimal and complete ligand descriptions. The
restraint library may contain two types of ligand descriptions:
minimal and complete (Vagin et al., 2004). Along with the
molecular graph, which incorporates lists of atoms and covalent bonds and their types, the minimal description contains
types of chiral centres and atom names. In addition to this
information, the complete description contains the target
values and uncertainties for template restraints (covalent
bond lengths and angles) and a list of planar groups. The
CCP4 program LibCheck can extend the minimal ligand
description to a complete description using the definitions of
atom types, which are also included in the restraint library.
Ligand descriptions (currently only complete descriptions) are
stored in the restraint library as files in CIF format (Hall et al.,
1991). The minimal description and eventually the complete
description can be derived from other molecular formats. In
particular, SDF (Dalby et al., 1992),
SYBYL mol2 (Tripos, 2005) and
PDB files, as well as SMILE strings
(Weininger, 1988; Greaves et al., 1999),
can be used as input files for LibCheck.
In addition to the complete ligand
description, LibCheck outputs the
Cartesian coordinates of the atoms that
satisfy geometrical restraints from the
complete description.
2.1.2. Creating a complete ligand
description. JLigand is a graphical
Figure 2
REFMAC checkpoints for refinement of glycosylated proteins. The figure shows fragments of (a) the
output PDB file and (b) the REFMAC log file after successful refinement of the structure presented
in Fig. 1. LINKR records indicate that REFMAC was able to associate short distances between
monomers with library link entries and corresponding warnings in the log file prompt the user to
check whether this assignment is correct and whether the linked atoms fit the electron density.
434
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JLigand
interface for LibCheck (or potentially
another similar application) that allows
the visualization and editing of the
minimal description and its transfer to
LibCheck for creation of the complete
description. Currently, JLigand can
only be launched from the command
line, but starting from CCP4 release
v.6.3.0 it will be possible to launch it
from the CCP4i interface. The set
of editing operations that JLigand
provides is typical of molecular editors,
i.e. adding a covalently bound atom or
a group, adding or deleting a bond or
changing the bond type, deleting the
atom and all of its bonds and changing
the atom type, name or, if applicable,
Acta Cryst. (2012). D68, 431–440
research papers
chirality type. Atomic coordinates are used to visualize the
molecular graph. Z coordinates of the atoms and groups to be
added are chosen in such a way that their addition takes place
in the plane that is parallel to the screen and goes through the
atom to which a new atom or group binds.
2.1.3. Regularization procedure in JLigand. In JLigand the
procedure of creating a complete ligand description is part of a
‘regularization’ procedure, which also includes the calculation
of the atomic coordinates that satisfy template restraints from
the complete description and are used in an updated threedimensional presentation of the ligand. Apart from having a
purely illustrative role, these coordinates can be saved as a
PDB file and then used, for example, in Coot to model this
ligand into the electron density. The complete description of
the regularized ligand can be saved into a new additional
library file or appended to an existing one.
A similar regularization procedure is carried out, albeit
implicitly, when ligands are loaded from the restraint library
or from external sources containing the molecular graph (SDF
or SMILE). By default, atomic coordinates and restraints, if
present in the input file, are ignored and only the molecular
graph, atom names and chiralities, if specified, are used. The
reason for such an approach is explained below (x2.3) and
relates to generation of link library entries.
The regularization procedure includes two steps. Firstly, the
information from the displayed ligand or from an input file is
saved as a CIF file with minimal description and loaded into
LibCheck, which generates a CIF file with complete description and a PDB file with atomic coordinates. Secondly, these
coordinates are refined using REFMAC in idealization mode,
with a CIF file being used as an additional library defining
target values of geometrical restraints. The refined coordinates
are then used to update or generate a graphical representation
of the ligand that is being regularized or imported.
2.1.4. Using coordinates for generation of ligand description. Another important option in LibCheck is the generation
of a complete ligand description from atomic coordinates (a
PDB file). In this case the minimal description is derived from
coordinates and the molecular graph may be incorrect for
coordinates with large uncertainty, although information on
atom types contained in the PDB file helps in assignment of
covalent bonds and their orders. With this option, chiralities
of atoms are also derived from the coordinates and once the
molecular graph has been generated and chiralities have been
assigned the input coordinates are discarded. The subsequent
procedure follows the same path as the generation of a
complete description from minimal description.
There is yet another option of utilizing the input coordinates available in LibCheck. By default, target values for
template restraints are derived from the restraint-library entry
describing atom energy types, but these values can also be
derived directly from the input coordinates. This option can be
used for high-precision coordinates obtained by X-ray crystallography of small molecules, calculated using precise force
fields or built using fragment libraries as in CORINA
(Sadowski et al., 1994) or PRODRG (Schüttelkopf & van
Aalten, 2004). Such an option is also available in JLigand, but
Acta Cryst. (2012). D68, 431–440
Table 1
Special amino acids most frequently used in the PDB as of July 2011 with
their names in the restraint library.
Frequency of occurrence was calculated by counting LINK records in the PDB
file header.
Threeletter
code
SEP
TPO
PTR
CSO†
KCX
LLP
CSD‡
CME
TYS
DAL
M3L
MLY
OCS
ABA
CSW‡
ALY
CSX†
TPQ
HIC
DVA
Compound name
No. of
PDB files
Total in
all PDB
files
Phosphoserine, O-phospho-l-serine
Phosphothreonine, O-phospho-l-threonine
Phosphotyrosine, O-phospho-l-tyrosine
Cysteine sulfenic acid, S-oxycysteine
Lysine NZ-carboxylic acid, NZ-formyllysine
N0 -Pyridoxyl-lysine-50 -monophosphate
Cysteine sulfinic acid
S,S-(2-Hydroxyethyl)thiocysteine
Tyrosine-O-sulfate, sulfonated tyrosine
d-Alanine
N-Trimethyllysine
N-Dimethyllysine
Cysteine sulfonic acid
-Aminobutyrate, -aminobutyric acid
Cysteine sulfinic acid
NZ-Acetyllysine
S-Oxycysteine, cysteine sulfenic acid
2,4,5-Trihydrophenylalanine quinone
4-Methylhistidine
d-Valine
381
326
270
254
229
221
167
150
140
122
118
115
99
83
73
70
63
53
50
50
757
590
517
531
573
448
330
554
161
315
165
4272
225
262
147
106
120
117
75
249
† The oxidized cysteine compound entries CSO and CSX (cysteine sulfenic acid) are
equivalent apart from the restraint library’s interpretation of the type of S—O bond
(single or double). ‡ The same applies to entries CSD and CSW for cysteine sulfinic
acid.
is not activated by default because it may result in modifications with too many edits (x2.2). To activate this possibility,
one needs to start JLigand from the command line with the
key ‘-as_is’. The menu element ‘File > Open As Is’ is then
activated that contains submenus for SDF, PDB and CIF
formats. CIF format is included because it corresponds to an
additional library file which may also contain coordinates.
2.2. Covalent linkages made by amino acids
2.2.1. Modified amino acids. Side chains of amino acids
can make covalent linkages with polysaccharides, nonpolymer
ligands, small groups or single atoms. Polysaccharides are
diverse branching polymers and restraints for covalent
linkages between their sugar monomers are most efficiently
described by a link and modification mechanism (x1.3). A
completely different mechanism is used to define restraints
for linkages between amino acids and nonpolymers or single
atoms. The whole compound including amino-acid and ligand
moieties is described in the library as a single monomer. This
monomer belongs to the group ‘peptide’ to ensure that it will
be automatically incorporated into a polypeptide chain during
refinement using generic links and modifications. There are
433 such special amino acids in the restraint library (the
library was last synchronized with the PDB Chemical
Components Dictionary at the end of 2010) and Table 1
presents the special amino acids most often encountered in the
PDB. As can be seen, 80% of the special amino acids in Table 1
can be obtained by the binding of a small group(s) with one
non-H atom to side-chain atom(s) of a ‘standard’ amino acid.
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Table 2
Describing the addition of one or a few atoms using link
library entries is not practical, but one can still describe
differences with a standard amino acid using a single modifiPairs contacting by atoms with names C and N are excluded. The indicated
cation entry which is not associated with any link entry.
pairs of atoms make close contacts in the range 1.14–2.22 Å in one or more
However, in many cases special amino acids contain two
PDB files. Fig. 3 presents two examples from this list, a relatively frequent one
distinct covalently bound moieties with an existing library
and a unique one, both corresponding to true covalent linkages. However, the
full list has not been validated and may contain artefacts.
description, one of which is a ‘standard’ amino acid. These
special amino acids can alternatively be described using one
No. of
Total in all
Atoms in PDB link records
PDB files
PDB files
link and two modification entries. There is one such example
in Table 1, LLP, which can be represented as a link between
TYR
CE2
HIS
NE2
27
52
TYR
CE2
MET
SD
18
35
Lys and PLP (see also x2.3).
TYR
CB
HIS
ND1
14
56
The advantage of the last approach is that the amino-acid,
TYR
CE1
TRP
CH2
14
27
ligand
and atom names do not change, which makes more
TYR
CE2
CYS
SG
7
14
TYR
C
SER
OG
5
6
biological sense. However, this advantage is at the price of the
TYR
CE1
GLN
CG
3
6
changes in the linked compounds being assumed to be localTYR
CE1
CYS
SG
3
4
ized around the covalent linkage between the monomers.
TYR
OH
TYR
CE2
1
6
TYR
CE1
HIS
NE2
1
4
Actually, both approaches are sufficiently accurate for correct
TYR
CE1
SER
O
1
4
interpretation of electron density in most X-ray experiments,
TYR
CZ
SER
C
1
4
including those at high resolution.
TYR
CZ
SER
N
1
4
TYR
CE1
ASP
N
1
3
2.2.2. Covalent links between amino acids. As described
All 52 combinations
135
274
previously, six links (four of which involve proline) and
corresponding modifications represent
all possible variations of the peptide
bond, a dominant covalent interaction
between amino acids in proteins.
However, there are many infrequently
occurring covalent linkages between
side chains or between the side and
main chains of two amino acids. These
linkages are quite diverse and corresponding template restraints are not
included in the restraint library. Importantly, description of such a linkage as a
special amino acid is not possible, as
such an amino acid would participate in
not two but four peptide bonds.
The covalent linkages making the
backbone of polypeptide or polynucleotide chains are not shown explicitly in the PDB coordinate files, while
all others are shown in LINK records.
The LINK records in the PDB entries
available on 17 July 2011 were parsed to
extract all special linkages between
amino acids. (LINK records should not
refer to C and N atoms of two amino
acids or two cysteine S atoms and
therefore all such cases were considered
to be mistakes and were excluded from
Figure 3
Examples of side chain to side chain and side chain to main chain covalent linkages in proteins (see
the analysis.) This search resulted in a
citations and PDB codes in the main text): (a) Tyr–Tyr covalent link in M. tuberculosis haemoglobin
total of 1279 examples distributed
O. (b) Tyr–His covalent link in HPII from E. coli. In both (a) and (b) the new covalent bond
among 766 PDB entries and belonging
increases the rigidity of the haem site. (c) Lys to main chain (C-terminal Gly) link between ubiquitin
to 571 different types of linkage. There
molecules in the complex of the TAB2 protein with diubiquitin. TAB2 only binds linked ubiquitin
molecules; the interaction is not shown, being distant from the link. (d) Covalent link between
were 365 unique examples, some of
ubiquitin main chain and the side chain of Ser from the ubiquitin-conjugation enzyme Ubc13. Lys63
which might be artefacts, and 161
of the second ubiquitin is poised to attack the intermediate ester bond. Residues involved in
examples which occur several times in a
covalent-linkage formation are shown in ball-and-stick representation. The linkages are shown in
single PDB entry. The most frequent
grey and are indicated by arrows.
Occurrences of Tyr paired with any amino acid in LINK records in the
PDB.
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nonpeptide linkages of Tyr with side and main chains of other
amino acids are listed in Table 2. Two of these examples are
shown in Fig. 3. In both cases the electron-density maps are
well defined for the side chains involved in the covalent
interactions. The link CE(TYR)–OH(TYR) is unique so far
and can only be found in PDB entry 1ngk (Milani et al., 2003)
for Mycobacterium tuberculosis haemoglobin O (Fig. 3a). It
was suggested (Milani et al., 2003) that this covalent link could
form in the presence of oxidative species and might have a
functional role in increasing the rigidity of the haem distal site.
Such a modified residue, named di-isodityrosine, had been
reported previously and proposed to promote insolubilization
of plant cell-wall extensins (Brady et al., 1996). Another
example is the linkage CB(TYR)–ND1(HIS), with Tyr being
involved in an interaction with Fe atoms coordinated by a
haem. Such a link occurs in several PDB entries (Table 2), but
all these are actually different crystal structures of the same
protein, catalase HPII from Escherichia coli. One of these
structures with PDB code 3p9p (Jha et al., 2011) is shown in
Fig. 3(b). The linkage was first identified by Bravo et al. (1997),
who suggested that this covalent bond contributes to stability
of the haem site. Similar to the previous case, the formation of
this unusual modification was attributed to the attack of redox
species.
A typical example of a side-chain to main-chain covalent
linkage is that made by the C-terminal residue of ubiquitin
(Gly76) to the side chain of Lys. There are at least 36 crystal
structures in the PDB in which such linkages occur (and are
listed in a LINK record). The maximal resolution obtained
for the structure of a ubiquitinated protein was 1.18 Å in the
crystal structure of the protein TAB2 in complex with diubiquitin (Sato et al., 2009; PDB entry 3a9j; Fig. 3c). TAB2
(transforming growth factor -activated kinase 1 binding
protein 2) specifically recognizes Lys63-linked polyubiquitin
chains and this recognition is involved in critical cell-signalling
pathways.
Even with ubiquitins there can be variations in the target
amino acid, with a unique example of a linkage between
ubiquitin and the OG atom of Ser from ubiquitin-conjugating
enzyme (Ubc13) being reported by Eddins et al. (2006) (PDB
entry 2gmi; Fig. 3d). In this work, a C87S mutant was designed
to capture an ester intermediate in which Gly96 from the
ubiquitin C-terminus was covalently linked to Ser87 of Ubc13.
This mimicked the formation of an intermediate Cys87(Ubc)–
Gly96(first, donor ubiquitin) thioester bond before it is
attacked by Lys63 of the next, acceptor ubiquitin and an
isopeptide bond is formed as a step in the process of ubiquitin
chain elongation. While this is a somewhat artificial situation,
engineered bonds such as this are to be expected in many
biological studies and this example highlights the importance
of an additional library with case-specific template restraints.
2.3. Generation of template restraints for a linked monomer
using JLigand
2.3.1. Generation of link description. For a long time, new
modifications and links were created using a text editor. The
Acta Cryst. (2012). D68, 431–440
need for a convenient graphical tool for this work was the
main incentive for the development of JLigand. The procedure of creating an amino acid–ligand link is presented in Fig. 4
using the example of a Schiff-base linkage between a lysine
residue and a pyridoxal phosphate (PLP) resulting in the
formation of an internal aldimine in aminotransferases (see,
for example, Rhee et al., 1997).
For a user, the procedure resembles conducting a chemical
reaction in silico. Descriptions of both LYS and PLP are
present in the restraint library. To make structure refinement
possible, it is necessary to create an additional library with
template restraints for the covalent linkage between LYS and
PLP. The user types the three-letter codes of the compounds
or performs a keyword search to load the compounds into
JLigand (Fig. 4a). In the next step, the user has to delete or
add non-H atoms. In this example, the O4A atom of PLP has
to be deleted as required for formation of the aldimine bond
(Fig. 4b). Finally, the covalent linkage and its bond order are
defined (Fig. 4c). After regularization, the new link can be
saved in a new user’s additional library (File > Save As Link;
Fig. 4d) or appended to an existing additional library (File >
Append As Link). Fig. 4(d) shows a graphical representation
of the final result and Fig. 4(e) presents the content of
the additional library listing required modifications to LYS
and PLP monomers and restraints associated with the
formation of a covalent linkage between the modified monomers.
An additional library thus obtained can be used for
refinement of the structure where PLP is covalently bound to
lysine. However, the generation of template restraints is only
part of the overall procedure of crystal structure refinement
including new ligands or links. The complete cycle typically
includes the following steps: (i) the structure without ligands is
refined, (ii) descriptions of ligands or links are generated, (iii)
the ligands are positioned in the difference electron-density
map and (iv) final refinement is performed for the model with
the ligand using an additional library with required template
restraints. The procedure is presented in detail in the JLigand
tutorial, which can be found in the collection of tutorials in the
Documentation section of the CCP4 web site.
2.3.2. Creating a link: conventions and potential pitfalls. In
the Lys–PLP example the standard library already contained
the monomer descriptions. However, either one of the
monomers or both of them can be created by the user and then
joined with a covalent linkage during the same editing session.
In such a case, it is assumed that the final version of the
monomer description is that obtained from the last successful
regularization. All changes made to the molecular graph after
the final regularization and prior to joining the monomers with
a link will be written into the additional library as a modification. A subsequent regularization of the linked monomers is
usually accompanied by deletion or addition of H atoms. All
such automatic changes are also added to the monomer
modifications.
When the link is saved (File > Save As Link) all relevant
data are stored: the descriptions of the link and two modifications and, if one or both monomers had been edited, the
Lebedev et al.
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descriptions of these monomers
as well. With such an approach
many operations are carried out
implicitly and do not distract the
user’s attention with technical
details of library organization.
However, the user still has to be
careful. To illustrate this point: in
the example discussed above
regularization of PLP after
deleting O4A would have led to a
new monomer description being
written into the additional library
under the same name (PLP). This
would have blocked the PLP
description from the standard
restraint library, rendering the
use of this additional library for
the refinement of a structure
containing both the internal aldimine and free PLP impossible (as,
for instance, in PDB entry 2x5d;
Oke et al., 2010).
Such problems are inevitable
when many operations are
performed implicitly. Therefore,
future versions of JLigand may
provide a more transparent,
although more sophisticated,
interface explicitly showing lists
of monomers, links and modifications present in the standard and
user’s libraries.
2.3.3. Algorithm for generating
links and modifications. As in the
Figure 4
Defining restraints for the covalent linkage Lys–PLP using JLigand. Snapshots show the state of the
JLigand interface after the following user actions: (a) loading monomers LYS and PLP from the restraint
library, (b) removing O4A from PLP, (c) connecting C4A (PLP) with NZ (LYS) and defining the bond
order and (d) regularization. The Save As Link menu has to be used to generate (e) an additional library
file containing the following five data blocks: list of modifications, list of links, modifications LYSmod1 and
PLPmod1 to be applied to LYS and PLP, respectively, and link LYS–PLP. The file header, tables of
restraints for LYSmod1 and all the tables for PLPmod1 are omitted, as indicated by lines filled by tildes. H
atoms are dealt with automatically. It is also possible to visualize them and handle them explicitly.
438
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JLigand
case of monomers, the generation
of template restraints and atomic
coordinates for linked ligands is
reduced to running LibCheck and
REFMAC. The first step is the
generation of the minimal
description, which includes all the
atoms and bonds inherited from
modified monomers plus the bond
that makes a covalent linkage. In
the internal representation all
atoms are identified by pointers
on the objects associated with
them, but representation in a CIF
file is based on atom names.
Therefore, in the regularization
step atoms in the composite
compound including two monomers are given unique names and
the renamed compound goes
through the LibCheck–REFMAC
procedure in the same way as is
Acta Cryst. (2012). D68, 431–440
research papers
performed for individual monomers (x2.1). The original atom
names are then restored in the regularized composite
compound. If a new H atom is added by LibCheck, the name
for this atom is assigned based on the original name of a nonH atom to which it is connected. The graphical representation
of the new composite compound is the same as that for the
monomers. However, editing functions are blocked (the
exception is the bond order of the covalent linkage between
the monomers). This is aimed at avoiding over-complicated
modifications.
On request (‘View’ or ‘Save’), but not before regularization,
the description of the total compound is split into two modifications and one link. Initially, the template restraints involving atoms from different monomers are written into the link
description and deleted from the description of the complete
molecule, which therefore splits into two modified monomers.
During editing, a reference to the parent atom from the initial
ligand is remembered for each atom of the edited ligand; such
a reference is missing for an atom added during editing.
Therefore, the lists of deleted and added atoms and restraints
can easily be restored after editing is finished. Besides, the
atoms and restraints which have not been added or removed
could have nevertheless changed. This is because the user
might have changed the type of the atom or bond, or the atom
and bond parameters might have changed during regularization because neighbouring atoms were added, deleted or
modified. In the current version of JLigand the changes in
atoms and bonds are traced by direct comparison of their old
and new parameters. If an alteration in the type of atom or
restraint has occurred or the changes in the target values of
restraints have exceeded the standard deviation declared in
the dictionary, the corresponding item is considered to be
modified and therefore is added to the modification data
block. Finally, the decision is made on the original monomers
to which modifications were applied. If these are available
from the main or additional restraint library their descriptions
are discarded. Otherwise, one or both monomers are added to
the additional library.
2.3.4. Drawbacks of the existing algorithm. If the
descriptions of the original monomers were generated by
another program or against a different set of atom types or
derived from atomic coordinates, then such an algorithm
would result in too many edits in the modification entries. This
is why by default all the imported monomer data are reduced
to a minimal description and the complete description is then
generated from scratch in the same manner as is performed
during the implicit generation of the description representing
linked monomers. This in particular is the reason why the
option ‘Read As Is’ is hidden by default and alternative
regularization protocols (using, for example, PRODRG for
generation of restraint parameters) have not yet been implemented. It is anticipated that in future versions of JLigand the
criterion of bond ‘alteration’ would be changed. The decision
on inclusion of the atom or restraint in the modification will
be based on changes in the molecular graph, e.g. defined by
whether any changes occurred in the first and second coordination spheres of the atom or restraint in question.
Acta Cryst. (2012). D68, 431–440
3. Conclusions
JLigand is a new CCP4 program for three-dimensional editing
of molecular graphs, the generation of corresponding template
restraints and their storage in the additional library file, which
can be used for model building and refinement together with
the standard restraint library. The molecular graph is generated from scratch or from the atomic coordinates (PDB file) or
imported from external sources such as a SMILE string, CIF
library file or SDF file and edited if necessary. The generation
of restraints from a molecular graph is performed using
LibCheck. Atomic coordinates are regularized using
REFMAC and are used to display the new molecule.
Two other types of entries that are present in the restraint
library and may also be present in the additional library are
modifications and links. 32 modifications from the standard
library are used for the generation of restraints for modified
monomers, while another 21 modifications and 65 links
describe changes and additional template restraints that are
necessary to assemble appropriate monomers into polypeptides, polynucleotides or polysaccharides. This small number
of standard library entries accounts for all or almost all of the
covalent linkages between monomers in a given structure,
while there could be a few novel linkages that are specific to a
limited number of structures. Even quite a superficial analysis
of the PDB showed that these specific linkages could be highly
diverse. Providing the graphical and computational tools
required for the generation of such descriptions is a distinctive
feature of JLigand.
The Java executable, source code and tutorials for JLigand5
will be available from CCP4 starting with v.6.3.0.
Part of this work was carried out while AAL, PY, AAV
and GNM were working in YSBL, Chemistry Department,
University of York supported by the BBSRC (grant No. BB/
F001134/1), Wellcome Trust (grant No. 064405/Z/01/A) and
CCP4. AAL is currently supported by CCP4 and GNM is
supported by MRC (MC_US_A025_0104).
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